The tumor microenvironment plays an important role in the processes of tumor growth, metastasis, and drug resistance. Three-dimensional (3D) tissue culture models have been used to mimic the tumor microenvironment in vivo. Imaging techniques are a powerful tool to analyze the key aspects of 3D tissue, including their size, growth, and volume, for example. Conventional imaging systems can provide high-throughput screening of 3D tumor cell cultures to improve the efficiency and cost-effectiveness of discovering potential tumor drug candidates. However, there is now increased evidence that the spatial resolution of conventional imaging is not sufficient to analyze cellular behaviors in 3D tumor cell culture.

Here we show new approaches to analyze cellular behaviors in 3D tumor cell culture based on the high-resolution images, captured by confocal laser scanning microscope. This 3D structural analysis in high resolution can reveal not only the ratio of tumor cells to stromal cells, but also the cell number of each in co-cultured tumor spheroids, efficacy of anticancer drugs in the cell population inside spheroids, which contain high number of cancer stem cells, 3D polarity of migration and invasion of tumor cells into the extracellular matrix that can be specifically targeted by anti-metastatic therapy, and dose-dependent drug-induced toxicity on mitochondria in 3D tumor cell cultures.

This microscopic 3D spheroid assay platform represents the first step toward the development of tools for understanding the pharmacological mechanisms of drugs or drug targets in preclinical cancer models under drug discovery. We hope that further development of this assay platform will allow the study of drug dosage, efficacy, and resistance, before preclinical and clinical studies.